This invention is in the field of spectrophotometric determinations of concentrations of analytes in samples. The invention further relates to methods of calibrating spectrophotometers. Particularly, the method relates to the calibration of spectrophotometric apparatus designed to measure interferents in serum and plasma.
Clinical laboratory tests are routinely performed on serum or plasma of whole blood. In a routine assay, red blood cells are separated from plasma by centrifugation. Red blood cells and various plasma proteins may also be separated from serum by clotting prior to centrifugation. Hemoglobin (Hb), light-scattering substances like lipid particles, bile pigments bilirubin (BR) and biliverdin (BV) are typical substances which will interfere with and affect spectrophotometric and other blood analytical measurements and are therefore referred to as interferents. The presence of such interferents affects the ability to perform tests on the serum or plasma and as such can be the to compromise specimen integrity.
Visual inspection can be used to determine the presence of interferents in serum and plasma but such a method relies on the experience and knowledge of the observer and is therefore unreliable. The use of an apparatus or instrument to measure interferents in serum and plasma i.e., assess specimen integrity, is a substitute for visual inspection and the interferents may be regarded as analytes with respect to the apparatus. Measurement of interferents is taught in WO 9838961 and WO 9839634. Because quantitative results from the determination of the concentration of such interferents are reported based on specific calibration algorithms, there is a need to calibrate and to monitor calibration performance daily.
Unlike many blood analytical apparatus, calibration of reagentless spectrophotometric apparatus used to measure the concentration of analytes or interferents in a serum or plasma sample is a cumbersome time intensive exercise. Each apparatus used for the purposes of determining the concentration of interferents must be calibrated according to procedures known in the art, for example, the process described herein, in the section titled xe2x80x9cPrimary Calibration,xe2x80x9d and over the lifetime of an apparatus can amount to a considerable amount of time and cost. Furthermore, in settings where a large number of apparatus is needed to perform multiple sample measurements (such as blood banks for example) the time required for calibration can become a real burden on the efficiency of the of the quality control process.
Martinek (J. Amer. Med. Technol., July-August 1978, p. 210-216) teaches a method of photometric correction, involving liquid absorbance standards to correct one spectrophotometer to match another using a slope and bias correction. This method may also be used for test methods that require reagents.
U.S. Pat. No. 4,866,644 teaches a method of calibrating a second apparatus to produce results for a test sample, as if the sample was tested on a first apparatus. The method combines photometric correction with a mathematical process that computes a waveshift for each index point. The waveshifts are derived from the assessment of readings determined for a plurality of samples on the two apparatus. The waveshifts are applied as corrections to an existing wavelength calibration table of the second apparatus in order to make the second apparatus behave in a manner similar to the first apparatus. In U.S. Pat. No. 4,866,644, the same wavelengths are assigned to the same corresponding index points in every instrument. Therefore, there is no derivation of a new wavelength calibration table of the second instrument, and the waveshift correction is applied to each measurement as it is determined on the second instrument.
Ozdemir, D et al (Applied Spectroscopy, Volume 52 No. 4, 1998, p599-603) described an alternative to calibration transfer, referred to as xe2x80x9cHybrid Calibration Models,xe2x80x9d which teaches the inclusion of calibration data obtained from more than one instruments, in developing primary calibration algorithms.
WO 94/08225 discloses a method involving the modification of the constants of a primary calibration algorithm of a second or recalibrated apparatus, to yield results consistent with a first apparatus that is in control. A limitation of this method is that the number of samples required must be at least one more than the number of terms used in the primary calibration equation, because a mathematical system of xe2x80x9csimultaneous equationsxe2x80x9d is used to generate a new constant for each term in the primary calibration algorithm. Furthermore, a predicted dependant variable, such as a chemical or physical property, of a calibrator is required to generate the new constants.
WO 97/47942 teaches a method for a second apparatus to produce results for a test sample, as if the sample was tested on the first apparatus involving testing a set of stable samples, whose absorbance spectra mimic that of the analytes, on both the first and a second apparatus, and predicting the analyte concentrations after applying a primary calibration algorithm. This method requires a predicted dependant variable, for example a chemical or physical property, of the calibrator. Analyte results predicted by both apparatus are used to perform a slope and bias correction of each analyte prediction on a test sample. The calibration set requires the property of having an absorbance spectra similar to the analyte.
There is a need for a method to simply and accurately calibrate a second apparatus, and to recalibrate a first or second apparatus that is no longer in control.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.
This invention is in the field of spectrophotometric determination of concentrations of analytes in samples. The invention further relates to methods of calibrating spectrophotometers. The method may be used for the calibration of spectrophotometric apparatus designed to measure interferents in serum and plasma. The invention also relates to a method of transferring calibration algorithms from a first apparatus to a second apparatus, with the optional use of data pre-processing techniques, and photometric correction.
The present inventor has found that for a given analyte, a xe2x80x9cPrimary Calibration Algorithmxe2x80x9d developed for a xe2x80x9cFirst apparatusxe2x80x9d can be transferred onto a xe2x80x9cSecond Apparatusxe2x80x9d. Therefore, the Second Apparatus need not be subjected to the cumbersome, time intensive Primary Calibration process.
In one aspect of the invention, the First Apparatus that is known to be xe2x80x9cin Controlxe2x80x9d is used to assign absorbance values to a xe2x80x9cSet of Calibratorsxe2x80x9d from a batch or lot, and any Second Apparatus can be calibrated rapidly by a process of xe2x80x9cCalibration Algorithm Transfer,xe2x80x9d and the concentration of an analyte in a sample determined by applying the xe2x80x9cPrimary Calibration Algorithmxe2x80x9d to a corrected interpolated absorbance measurement of the sample. Therefore, the present invention provides a method for calibrating a Second Apparatus using a Set of Calibrators with absorbances assigned by the First Apparatus.
In yet a further aspect of the invention a method for adjusting the absorbance of sample obtained on a second apparatus to normalize it with that of a first apparatus that is in control (xe2x80x9cphotometric correctionxe2x80x9d) using a xe2x80x9cLinear Regression Equationxe2x80x9d is also provided.
The present invention provides a method (A) of determining the concentration of one or more Analytes in a Sample in a second apparatus comprising:
(i) incorporating at least one primary calibration algorithm that uses an order derivative of absorbance obtained for at least one of a standard set of wavelengths, on the second apparatus;
(ii) measuring absorbance values of the sample at one or more than one wavelength from the standard set of wavelengths on the second apparatus;
(iii) obtaining the order derivative of the absorbance values;
a) if the order derivative is not zero, then using the order derivative and calculating a concentration of the Analyte in the sample, by applying the Primary Calibration Algorithm to the order derivative of absorbance obtained;
b) if the order derivative is zero, then calculating a concentration of the Analyte in the sample, by applying the Primary Calibration Algorithm to the absorbance values.
The present invention pertains to the method (A) defined above, wherein in the step of incorporating (step (i)), and in the step of obtaining (step (iii)), the order derivative, is of a zero, first, second, or third order. Furthermore, a statistical technique selected from the group consisting of simple linear regression, multiple linear regression, and Multivariate data analysis, may be used to process absorbance measurements for the development of the at least one primary calibration algorithm, the Multivariate data analysis selected from the group consisting of Principal Component Analysis, Principal Component Regression, Partial Least Squares regression, and Neural Network. Also, the present invention provides a step of data pre-processing following the step of measuring (step ii)), wherein data pre-processing is selected from the group consisting of calculation of interpolated absorbances; smoothing of absorbances; calculation of a first and higher order derivative of absorbance; multiplicative scatter correction; data transformation; photometric correction, and any combination thereof.
The present invention provides the method (A), above, wherein the second apparatus comprises a second linear diode array comprising the same number of pixels as a first linear diode array in a first apparatus.
The present invention embraces the method (A) above, wherein in the step of incorporating (step (i)), and wherein in the step of measuring (step (ii)), the standard set of wavelengths is a set of approximate wavelengths derived from a wavelength calibration table of a first apparatus, the second apparatus, or both a first and the second apparatus.
The present invention pertains to the method (A) above, wherein in the step of incorporating (step (i)), and wherein in the step of measuring (step (ii)), the standard set of wavelengths comprises wavelengths that are common to a wavelength calibration table of both a first apparatus used to obtain the primary calibration algorithm, and the second apparatus.
This invention also includes the method (A) above, wherein in the step of incorporating (step (i)), the at least one primary calibration algorithm is developed from a combination of measurements obtained from one or more primary calibrators measured on a first apparatus and one or more similar apparatus.
The present invention also pertains to the method (A), above, wherein the standard set of wavelengths comprises wavelengths from about 300 nm to about 2500 nm, or wherein the standard set of wavelengths comprises wavelengths from about 500 nm to about 1100 nm.
The present invention embraces the method (A) above, wherein, in the step of measuring (step ii)), Photometric Correction is performed.
The present invention also provides the method (A) above, wherein in the steps of incorporating (step (i)), and measuring (step (ii)), the standard set of wavelengths is obtained by creating a table of approximate wavelengths derived from a first, a second or both the first and the second wavelength calibration table, wherein a pixel number of a linear diode array of the first apparatus and a pixel number of a second linear diode array of the second apparatus must be within less than or equal to about xc2x1N pixel of a reference pixel number of the first apparatus, where, N is a number of pixels that encompass a range of wavelengths of no more than about xc2x120 nm, wherein the reference pixel number of the first apparatus is associated with a known wavelength of electromagnetic radiation.
The present invention embraces the method (A), above, wherein the wavelength calibration table for the first apparatus or the second apparatus is obtained by:
(i) projecting a first electromagnetic radiation of known wavelength, onto a first pixel of a first linear diode array of the first apparatus, or a second linear diode array of the second apparatus;
(ii) using a second electromagnetic radiation of known wavelength, the second electromagnetic radiation having a different wavelength than the first electromagnetic radiation, projecting the second electromagnetic radiation onto a second pixel of the first or the second linear diode array;
(iii) identifying the first and second pixels within the first or the second linear diode array;
(iv) calculating a pixeldispersion for the first or the second linear diode array; and
(v) assigning a wavelength to each pixel within the first or the second linear diode array to produce the wavelength calibration table using the pixeldispersion and either the first electromagnetic radiation of known wavelength and the first pixel, or the second electromagnetic radiation of known wavelength and the second pixel.
Also included within the present invention is the method (A), above, wherein the wavelength calibration table for the first or the second apparatus is obtained by:
(a) projecting a known wavelength of electromagnetic radiation, onto a pixel in a linear diode array of the first apparatus, or the second apparatus;
(b) identifying pixel number of the pixel;
(c) assigning a wavelength to each pixel within the linear diode array to produce the first, second, or both the first and the second wavelength calibration table using a predetermined pixeldispersion, the known wavelength of electromagnetic radiation and the pixel number.
The present invention also provides a method (B) of determining the concentration of one or more Analytes in a Sample in a second apparatus comprising:
(i) incorporating at least one primary calibration algorithm that uses an order derivative of absorbance obtained for at least one of a standard set of wavelengths, on the second apparatus, where wavelengths of the standard set of wavelengths are the same as wavelengths of a wavelength calibration table on the second apparatus;
(ii) measuring absorbance values of the sample at one or more wavelengths from the standard set of wavelengths, on the second apparatus;
(iii) obtaining the order derivative of the absorbance values;
a) if the order derivative is not zero, then using the order derivative and calculating a concentration of the Analyte in the sample, by applying the Primary Calibration Algorithm to the order derivative of absorbance obtained;
b) if the order derivative is zero, then calculating a concentration of the Analyte in the sample, by applying the Primary Calibration Algorithm to the absorbance values.
This invention embraces the method (B) above, wherein in the step of incorporating (step (i)), the at least one primary calibration algorithm is developed from a combination of measurements obtained from one or more primary calibrators measured on a first apparatus and one or more similar apparatus.
The present invention also includes the method (B), wherein in the step of incorporating (step (i)), and in the step of obtaining (step (iii)), the order derivative, is of a zero, first, second, or third order.
The present invention also pertains to the method (B), above, wherein a statistical technique selected from the group consisting of simple linear regression, multiple linear regression, and Multivariate data analysis, is used to process absorbance measurements for the development of the at least one primary calibration algorithm, and wherein the Multivariate data analysis is selected from the group consisting of Principal Component Analysis, Principal Component Regression, Partial Least Squares regression, and Neural Network. Furthermore, a step of data pre-processing may follow the step of measuring (step ii)) of Method (B), wherein data preprocessing is selected from the group consisting of calculation of interpolated absorbances; smoothing of absorbances; calculation of a first and higher order derivative of absorbance; multiplicative scatter correction; data transformation; photometric correction, and any combination thereof.
The present invention embraces the method (B) above, wherein, in the step of measuring (step ii)), Photometric Correction is performed.
The present invention also pertains to the method (B), above, wherein in the steps of incorporating (step (i)), and measuring (step (ii)), the standard set of wavelengths is obtained by creating a table of approximate wavelengths derived from a first, a second or both the first and the second wavelength calibration table, wherein a pixel number of a linear diode array of the first apparatus and a pixel number of a second linear diode array of the second apparatus must be within less than or equal to about xc2x1N pixel of a reference pixel number of the first apparatus, where, N is a number of pixels that encompass a range of wavelengths of no more than about xc2x120 nm, wherein the reference pixel number of the first apparatus is associated with a known wavelength of electromagnetic radiation.
The present invention provides the method (B), above, wherein the wavelength calibration table for the first apparatus or the second apparatus is obtained by:
(i) projecting a first electromagnetic radiation of known wavelength, onto a first pixel of a first linear diode array of the first apparatus, or a second linear diode array of the second apparatus;
(ii) using a second electromagnetic radiation of known wavelength, the second electromagnetic radiation having a different wavelength than the first electromagnetic radiation, projecting the second electromagnetic radiation onto a second pixel of the first or the second linear diode array;
(iii) identifying the first and second pixels within the first or the second linear diode array;
(iv) calculating a pixeldispersion for the first or the second linear diode array; and
(v) assigning a wavelength to each pixel within the first or the second linear diode array to produce the wavelength calibration table using the pixeldispersion and either the first electromagnetic radiation of known wavelength and the first pixel, or the second electromagnetic radiation of known wavelength and the second pixel.
The present invention also pertains to the method (B), above, wherein the wavelength calibration table for the first or the second apparatus is obtained by:
(a) projecting a known wavelength of electromagnetic radiation, onto a pixel in a linear diode array of the first apparatus, or the second apparatus;
(b) identifying pixel number of the pixel;
(c) assigning a wavelength to each pixel within the linear diode array to produce the first, second, or both the first and the second wavelength calibration table using a predetermined pixeldispersion, the known wavelength of electromagnetic radiation and the pixel number.
The present invention also provides a method (C), of determining the concentration of at least one Analyte in a Sample in a second apparatus comprising:
(i) incorporating at least one upgraded primary calibration algorithm on the second apparatus, the at least one upgraded primary calibration algorithm developed by combining an original primary calibration data set obtained from one or more first apparatus for at least one of a standard set of wavelengths, with additional data from the second apparatus, the additional data obtained using a smaller similar primary calibration set, a subset primary calibration set, or both, for at least one of a standard set of wavelengths;
(ii) measuring absorbance values of the sample at one or more wavelengths from the standard set of wavelengths, on the second apparatus;
(iii) obtaining the order derivative of the absorbance values;
a) if the order derivative is not zero, then using the order derivative and calculating a concentration of the Analyte in the sample, by applying the Primary Calibration Algorithm to the order derivative obtained;
b) if the order derivative is zero, then calculating a concentration of the Analyte in the sample, by applying the Primary Calibration Algorithm to the absorbance values.
The present invention embraces the method (C), above, wherein in the step of incorporating (step (i)), and in the step of obtaining (step (iii)), the order derivative, is of a zero, first, second, or third order.
The present invention also pertains to the method (C), above, wherein a statistical technique selected from the group consisting of simple linear regression, multiple linear regression, and Multivariate data analysis, is used to process absorbance measurements for the development of the at least one primary calibration algorithm, and wherein the Multivariate data analysis is selected from the group consisting of Principal Component Analysis, Principal Component Regression, Partial Least Squares regression, and Neural Network. Furthermore, a step of data pre-processing may follow the step of measuring (step ii)) of Method (B), wherein data preprocessing is selected from the group consisting of calculation of interpolated absorbances; smoothing of absorbances; calculation of a first and higher order derivative of absorbance; multiplicative scatter correction; data transformation; photometric correction, and any combination thereof.
The present invention embraces the method (C) above, wherein, in the step of measuring (step ii)), Photometric Correction is performed.
The present invention also pertains to the method (C), above, wherein in the steps of incorporating (step (i)), and measuring (step (ii)), the standard set of wavelengths is obtained by creating a table of approximate wavelengths derived from a first, a second or both the first and the second wavelength calibration table, wherein a pixel number of a linear diode array of the first apparatus and a pixel number of a second linear diode array of the second apparatus must be within less than or equal to about xc2x1N pixel of a reference pixel number of the first apparatus, where, N is a number of pixels that encompass a range of wavelengths of no more than about xc2x120 nm, wherein the reference pixel number of the first apparatus is associated with a known wavelength of electromagnetic radiation.
The present invention provides the method (C), above, wherein the wavelength calibration table for the first apparatus or the second apparatus is obtained by:
(i) projecting a first electromagnetic radiation of known wavelength, onto a first pixel of a first linear diode array of the first apparatus, or a second linear diode array of the second apparatus;
(ii) using a second electromagnetic radiation of known wavelength, the second electromagnetic radiation having a different wavelength than the first electromagnetic radiation, projecting the second electromagnetic radiation onto a second pixel of the first or the second linear diode array;
(iii) identifying the first and second pixels within the first or the second linear diode array;
(iv) calculating a pixeldispersion for the first or the second linear diode array; and
(v) assigning a wavelength to each pixel within the first or the second linear diode array to produce the wavelength calibration table using the pixeldispersion and either the first electromagnetic radiation of known wavelength and the first pixel, or the second electromagnetic radiation of known wavelength and the second pixel.
The present invention also provides the method (C) above, wherein the wavelength calibration table for the first or the second apparatus is obtained by:
(a) projecting a known wavelength of electromagnetic radiation, onto a pixel in a linear diode array of the first apparatus, or the second apparatus;
(b) identifying pixel number of the pixel;
(c) assigning a wavelength to each pixel within the linear diode array to produce the first, second, or both the first and the second wavelength calibration table using a predetermined pixeldispersion, the known wavelength of electromagnetic radiation and the pixel number.
The present invention also includes the method (D) above, wherein a statistical technique selected from the group consisting of simple linear regression, multiple linear regression, and Multivariate data analysis, is used to process absorbance measurements for the development of the at least one primary calibration algorithm, wherein the Multivariate data analysis is selected from the group consisting of Principal Component Analysis, Principal Component Regression, Partial Least Squares regression, and Neural Network.
The present invention also provides the method (D), above, wherein in the steps of incorporating (step (i)), and measuring (step (ii)), the standard set of wavelengths is obtained by creating a table of approximate wavelengths derived from a first, a second or both the first and the second wavelength calibration table, wherein a pixel number of a linear diode array of the first apparatus and a pixel number of a second linear diode array of the second apparatus must be within less than or equal to about xc2x1N pixel of a reference pixel number of the first apparatus, where, N is a number of pixels that encompass a range of wavelengths of no more than about xc2x120 nm, wherein the reference pixel number of the first apparatus is associated with a known wavelength of electromagnetic radiation.
The present invention pertains to the method (D), above, wherein the wavelength calibration table for the first apparatus or the second apparatus is obtained by:
(i) projecting a first electromagnetic radiation of known wavelength, onto a first pixel of a first linear diode array of the first apparatus, or a second linear diode array of the second apparatus;
(ii) using a second electromagnetic radiation of known wavelength, the second electromagnetic radiation having a different wavelength than the first electromagnetic radiation, projecting the second electromagnetic radiation onto a second pixel of the first or the second linear diode array;
(iii) identifying the first and second pixels within the first or the second linear diode array;
(iv) calculating a pixeldispersion for the first or the second linear diode array; and
(v) assigning a wavelength to each pixel within the first or the second linear diode array to produce the wavelength calibration table using the pixeldispersion and either the first electromagnetic radiation of known wavelength and the first pixel, or the second electromagnetic radiation of known wavelength and the second pixel.
The present invention also provides the method (D), above, wherein the wavelength calibration table for the first or the second apparatus is obtained by:
(a) projecting a known wavelength of electromagnetic radiation, onto a pixel in a linear diode array of the first apparatus, or the second apparatus;
(b) identifying pixel number of the pixel;
(c) assigning a wavelength to each pixel within the linear diode array to produce the first, second, or both the first and the second wavelength calibration table using a predetermined pixeldispersion, the known wavelength of electromagnetic radiation and the pixel number.
In a further aspect of the invention, a Second Apparatus that was calibrated by xe2x80x9cCalibration Algorithm Transferxe2x80x9d but is no longer in control, can be xe2x80x9cRecalibratedxe2x80x9d using a Set of Calibrators that was assigned absorbances or absorbance values by the First Apparatus, which was known to be in control. The present invention also provides a method for Recalibration of the First Apparatus in the same way as any Second Apparatus.
The inventor has also found that the process of Calibration Algorithm Transfer and subsequent determination of analyte concentration can be accomplished by using an order of derivative of the absorbance in the Primary Calibration Algorithm, where absorbance correction or xe2x80x9cPhotometric Correctionxe2x80x9d may not be necessary, provided that the order of derivative of absorbance used in the Primary Calibration Algorithm at the selected wavelength(s) does not contain significant inter-apparatus variability as may be seen in the absorbances at the same wavelength(s). Absorbance variability between apparatus can be minimized in certain xe2x80x9cSections of the Absorbance Spectra,xe2x80x9d by using an order of derivative of the absorbance.
The present invention provides a method to provide a simple reliable method of using primary calibration algorithms in a second apparatus that do not require representative samples for which the apparatus was designed. Rather, the standard samples used to calibrate a second apparatus can be any stable samples that produce a range of absorbances at all relevant wavelengths.
The prior art teaches how to calibrate a first apparatus using a derivative of absorbance, but the inventor is not aware of any prior art that teaches how to obtain an analyte concentration from a second apparatus, by using a calibration algorithm derived from the derivative of absorbances, and obtained on the first apparatus.
This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.